Since common epithelial cancers are resistant to most therapeutic treatments, much hope has been placed on prevention of cancer through the use of chemical agents or dietary manipulation. The widely used nonsteroidal antiinflammatory drugs (NSAIDs) are effective in chemoprevention of colorectal cancer, which is the second leading cause of cancer related death in the United States. However, the mechanisms underlying the anti-neoplastic effects of NSAIDs remain unclear. Substantial evidence has indicated that the chemopreventive activities of NSAIDs are mediated by induction of apoptosis. Our recent preliminary studies demonstrated that SMAC, a mitochondrial apoptogenic protein, plays an essential role in NSAID- induced apoptosis in colon cancer cells. SMAC is consistently released from the mitochondria into the cytosol during NSAID-induced apoptosis. Deletion of SMAC by homologous recombination or knockdown of SMAC by RNA interference in these cells abrogates NSAID-induced caspase activation and apoptosis. Furthermore, activation of SMAC and agents that mimic the function of SMAC can enhance the apoptotic response to sulindac, an NSAID commonly used in chemoprevention. These observations led us to hypothesize that SMAC mediates the anti-neoplastic effects of NSAIDs by promoting apoptotic cell death. We propose to test this hypothesis in vitro and in vivo using a combination of biochemical and genetic approaches. In the Aim 1, we will delineate the mechanisms by which NSAIDs induce SMAC release and apoptosis in colon cancer cells. We will focus on studying how NSAIDs modulate Bax and Bcl-XL to cause SMAC release and apoptosis. In the Aim 2, we will use cell culture and APCMM+ mouse models to determine if SMAC and SMAC mimetics sensitize the effects of NSAIDs in vitro and in vivo. In the Aim 3, we will use cell culture and xenograft tumor models to investigate if active SMAC and SMAC mimetics can restore NSAID sensitivity in NSAID-resistant colon cancer cells in vitro and in vivo. Finally, we will determine in the Aim 4 if SMAC is necessary for sulindac-mediated chemoprevention, and if other mitochondrial apoptogenic proteins mediate the effects of sulindac. The proposed studies will provide new insights into the anti-neoplastic effects of NSAIDs. In the long run, the results of these studies could be useful for developing improved strategies and agents for chemoprevention of colon cancer.